Despite their low natural abundance, rare disaccharides hold enormous potential for practical application.
Sucrose (table sugar) is the most widely used sweetener in food production due to its physiochemical and sensorial characteristics, however in recent decades an increased studies on alternative sweeteners is progressing rapidly, since of its high caloric value. Research on the production of oligosaccharides for foods with health benefits was started around late 1970s, and several oligosaccharides such as starch-related, sucrose-related, and lactose-related oligosaccharides have been developed.
Artificial sweeteners also called sugar substitutes were developed in recent years and are used in remarkable amounts instead of sucrose to sweeten foods and beverages but also in drugs and sanitary products. These sweeteners are not decomposed as carbohydrates and are not metabolized like sugars or only fermented slightly by the mouth microflora, which develops an artificial, metallic or licorice-like aftertaste. Therefore they often can be found blended in food to overcome this disadvantage.
Reducing disaccharides such as trehalulose (α-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (α-D-glucosylpyranosyl-1,6-Dfructofuranose) are structural isomers of sucrose (α-Dglucosylpyranosyl-1,2-β-D-fructofuranoside) and are naturally present in honey as well as in sugar cane extract in very low quantities. These natural sugars display a sweetening power, bulk, organoleptic and physical properties similar to those of sucrose.
Trehalulose is a non-crystalline saccharide which readily dissolves in water, does not substantially have cariogenicity, and has an about 70% sweetening power of sucrose, it is greatly expected to be used in food products, especially in foods enriched with sweeteners.
Isomaltulose is suitable sucrose replacement, since it's approximately half as sweet as sucrose, and has a similar sweetness quality and has non-carcinogenic properties. Moreover isomaltulose is also used as a raw material for production of another sweetener namely isomalt (palatint) by hydrogenation. Furthermore the reducing property of isomaltulose makes it an attractive industrial precursor for the manufacturing of biosurfactants and biocompatible polymers.
Both disaccharides: isomaltulose and trehalulose can be produced at an industrial scale through isomerization of sucrose using Sucrose Isomerases (SIase) which bio-convert sucrose into both isomaltulose and trehalulose with trace amounts of glucose and fructose.
U.S. Pat. No. 5,336,617 relates to a process for preparing trehalulose and isomaltulose wherein at least the trehalulose-forming enzyme system of a trehalulose-forming microorganism is contacted with a sucrose solution to convert it into trehalulose and isomaltulose in the weight ratio of at least 4:1
Commercial isomaltulose is produced from food grade sucrose through enzymatic isomerisation with sucrose-6-glucosylmutase. In this process the biocatalyst used to convert the sucrose is obtained from non-viable, non-pathogenic P. rubrum (CBS 574.44) cells which were killed using formaldehyde before they are added to the sucrose (U.S. Pat. No. 4,640,894 and U.S. Pat. No. 5,336,617).
The enzymes responsible for bioconversion of sucrose in to its corresponding isomers (Sucrose to isomaltulose or trehalulose) have been reported from different microorganisms such as Protaminobacter rubrum, Serratia plymuthica, Erwinia rhaponiciti for isomaltulose production whereas trehalulose producing organisms are Pseudomonas mesoacidophila and Agrobacterium raadiobacter. 
U.S. Pat. No. 4,857,461 discloses the process of extraction of sucrose mutase from periplasmic membranes of Protaminobacter rubrum or Serratia plymuthica and utilization of same in a radial type bioreactor for the conversion of sucrose to isomaltulose.
In non-patent literature L. Wu et. al., 2004 and 2005 identified a sucrose isomerase from Pantoea dispersa UQ68J having isomaltulose synthase activity and expressed in E. coli. However the recombinant isomaltulose synthase expressed in E. coli carried an additional carboxy-terminal six-His tag and moreover the expression level was low. Similarly, Nagai et al., 1994 showed that sucrose isomerase (also called as trehalulose synthase) activity of the P mesoacidophila MX-45. Latter Watzlawick H et. al., 2009 cloned the gene for expression in E. coli. However both authors didn't disclose their expression level of sucrose isomerase which is critical for mass production of enzymes at industrial scale.
The mass production of pure trehulose and/or isomaltulose is critical to meet the commercial value due to insufficient production of enzymes as biocatalysts. Thus mass production of sucrose isomerase and optimized bioconversion and downstream process is essential to make predominant product of trehulose and/or isomaltulose. Therefore heterologus expression of such enzymes is extremely desired to design a cost effective and much safe bioconversion process. Heterologus expression of gene products in different expression system is sometimes limited by the presence of codons that are infrequently used in other organisms. Expression of such genes can be enhanced by systematic substitution of the endogenous codons with codons over represented in highly expressed prokaryotic genes. Redesigning a naturally occurring gene sequence by choosing different codons without necessarily altering the encoded amino acid sequence often dramatically increased protein expression levels. One disadvantage in biocatalyst used in production of low caloric sugar such as isomaltulose or trehalulose are the production cost of the enzyme due to low expression level of enzymes in native or heterologus organisms which remains quite challenging.
Although number of microorganisms such as Protaminobacter rubrum, Serratia plymuthica, Erwinia rhaponiciti, Pseudomonas mesoacidophila and Agrobacterium raadiobacter have been recognized for their ability to produce sucrose isomerase to convert sucrose in to isomaltulose and/or trehalulose at different ratios in given reaction conditions. However the mass production of pure isomaltulose and/or trehalulose is critical to meet the commercial value due to insufficient production of enzyme as biocatalysts.
Even though the enzymes are known that are capable of catalyzing the rare sugars but the gap still remain in mass production of enzymes and difficulties in their expression levels.
The inventors has identified the production constrain of rare disaccharides which is a bottleneck for industrial scaling up and identified the expression level in heterologous is low for certain nucleotide which are less preferred. In order to overcome such problem, the nucleotide sequence obtained from Pseudomonas mesoacidophila MX45 and Pantoea dispersa UQ68J, encoding for the enzymes responsible for bio-conversion were modified to increase the expression level substantially. Such modification resulted in better expression of the enzymes sucrose isomerase of Pseudomonas mesoacidophila MX45 and isomaltulose synthase of Pantoea dispersa UQ68J in E. coli. The E. coli host organism used in the invention is JM109 (a K-12 E. coli strain) was used for heterologus expression of recombinant Sucrose isomerase and isomaltulose synthase. It has been shown the E. coli K-12 cannot be converted into an epidemic pathogen by laboratory manipulation with r-DNA molecules and it will not colonize the human intestinal tract.
The present invention offers an alternative process for producing rare disaccharides, in which the enzymes were expressed in E. coli at a higher level by modifying the gene sequence. In other words, the present research has made and effort to genetically modify the gene responsible for the production of enzymes, namely sucrose isomerase and isomaltulose synthase to be used in bioconversion of sucrose in to trehalulose and isomaltulose, respectively. The genetic modification has resulted in increase in expression of protein in E. coli host.